The Senior Investigator moved to the National Cancer Institute during FY2011 and hired personnel to set up a research laboratory during the first half of FY2012. A postdoctoral trainee was recruited to work on this RMS amplification project starting in March 2012. The first RMS genomic amplification studies are focusing on the 12q13-q14 amplicon, which occurs preferentially in a subset of PAX3-FOXO1-positive RMS (25% of cases) and smaller numbers of PAX7-FOXO1-positivie and fusion-negative cases. Our previous studies localized the minimal region of amplification to a 0.55 Mb region containing 28 genes, including the CDK4 proto-oncogene. Our subsequent microarray studies showed that 7 of these genes were consistently overexpressed at the RNA level in amplified RMS tumors. Collaborative studies with Dr. Javed Khan of the Pediatric Oncology branch applying high density array analysis and next generation sequencing is further refining these genomic findings.To determine which of these genes are overexpressed at the protein level, we set up a collaboration with Dr. Svetlana Pack and Dr. Stephen Hewitt of the Laboratory of Pathology, and Dr. Bruce Pawel of the Children's Hospital of Philadelphia (CHOP). Dr. Pawel has generated a tissue microarray of RMS specimens. In addition, two additional RMS TMA's were obtained from the Children's Oncology Group (COG). Previous work in my laboratory has determined the fusion status of most of the RMS specimens on both the CHOP and COG TMA's. Dr. Pack is developing a set of fluorescence in situ hybridization probes to determine the amplification status of the 12q13-q14 region in the cases on these TMA's. Dr. Hewitt and Dr. Pawel will then work with my laboratory to analyze the expression status of selected proteins n these TMA's by immunohistochemistry. To begin the functional analysis, we are initially focusing on the CDK4 gene and then will consider additional genes as indicated by our other analyses. For these studies, we have collected several RMS cell lines with the 12q13-q14 amplicon - Rh30 (PAX3-FOXO1-positive), 30SJ and 18C (fusion-negative) and HS-RMS-2 (pleomorphic). We have a series of RMS cell lines, both fusion-positive and negative, that lack the 12q13-q14 amplicon and will use selected lines in our studies. In addition to analyzing these cell lines in culture, our goal is to also develop a series of intramuscular xenografts from these cell lines. Towards this end, a subset of amplification positive and amplification negative lines is being tested for microbial pathogen contamination, and an animal studies protocol is now under review by the Institutional Animal Care and Use Committee. These animal studies are being planned in collaboration with Dr. Chand Khanna of the Pediatric Oncology Branch. Reagents are now being tested and validated for the CDK4 experiments. An antibody that detects CDK4 protein expression by western blot has been identified and successfully used in several experiments. Western blot analysis with this antibody confirms increased expression of CDK4 in lines with 12q13-q14 amplification. An initial retroviral shRNA expression construct targeted against the CDK4 gene was tested. Transfer into Rh30 cells demonstrated evidence of decreased CDK4 protein expression. Furthermore, cell culture studies indicate that the CDK4-shRNA-expressing cells grow more slowly than control cells. In complementary studies, a tetracycline-inducible lentiviral expression vector was provided by Dr. Ji Luo of the Medical Oncology Branch. We have subcloned the CDK4 cDNA into this expression vector and transduced the expression construct into Rh28 cells, which do not have amplification of the 12q13-q14 region. Initial dose-response and time course studies in Rh28 cells confirm that increased CDK4 protein expression is readily induced in transduced Rh28 cells in comparison to controls. Therefore, many of the basic techniques are now in place to investigate the role of CDK4 amplification and overexpression in rhabdomyosarcoma.